Chitosan induces Ca²⁺ -mediated programmed cell death in soybean cells

•  Chitosan, a component of the cell wall of many fungi, has been widely used to mimic pathogen attack and has been shown to induce several defence responses. •  Here we show that low concentrations (50 µg ml^-1 ) of chitosan are able to induce an increase in cytosolic Ca²⁺ concentration ([Ca²⁺ ]_cyt ), accumulation of H₂ O₂ in the culture medium, induction of the defence gene chalcone synthase (chs), and cell death in soybean cells (Glycine max). •  Chitosan-induced cell death occurred through cytoplasmic shrinkage, chromatin condensation and activation of caspase 3-like protease, suggesting the activation of a programmed cell death (PCD) pathway. Buffering extracellular Ca²⁺ with the Ca²⁺ chelator EGTA prevents [Ca²⁺ ]_cyt elevation, H₂ O₂ production and all downstream PCD features, but not cell death. •  Higher doses (200 µg ml^-1 ) of chitosan evoked neither Ca²⁺ transient and H₂ O₂ production nor caspase 3-like activation, but caused cell death, possibly as a result of plasma membrane disturbance.